Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor

ABSTRACT

A color-developing solution for use in automatic amino acid analysis by liquid chromatography, which comprises ninhydrin, an organic solvent, a buffer and ascorbic acid as a reducing agent for ninhydrin in an amount of from 0.2 X 10 3 to 0.5 X 10 3 mole per liter of solution.

United States Patent 1191 Arikawa et al.

145 Dec. 11, 1973 METHOD OF AUTOMATICALLY ANALYZING AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLOR-DEVELOPING SOLUTION THEREFOR [76] Inventors: Yoshijiro Arikawa, 3-20-22 Ose-cho; Seiji Takeuchi, 3-13-1 Ose-cho, both of Hitachi; Hisayuki Sagusa, 1030, lchige, Katsuta, all of Japan 22 Filed: Dec. 27, 1971 211 Appl. No.: 212,197

[30] Foreign Application Priority Data Dec. 25, 1970 Japan 45/117605 [52] U.S. Cl 23/230 M, 23/230 R [51] Int. Cl. G0ln 31/22 [58] Field of Search 23/230 M, 230 R; 252/408 [56] References Cited UNITED STATES PATENTS 3,230,048 l/l966 Skeggs 23/230 B X 3,506,403 4/1970 Fryer ct al. 23/230 R OTHER PUBLICATIONS Stathis, E. C., Anal. Chem. 20, 271 (1948).

Rakshit et al., Chem. Abstr. 52, 55011 (1958).

Merck Index, 7th Edn. 1960, p. 106.

Nakamura, 5., Chem. Abstr. 59, 4474f (1963).

Pako Corp., Chem. Abstr. 67, 49032j (1967) Kataoka et al., Chem. Abstr. 67, l6530s (1967).

Primary Examiner-Robert M. Reese AttorneyCraig, Antonelli & Hill [57] ABSTRACT A color-developing solution for use in automatic amino acid analysis by liquid chromatography, which comprises ninhydrin, an organic solvent, a buffer and ascorbic acid as a reducing agent for ninhydrin in an amount of from 0.2 X 10 to 0.5 X 10 mole per liter of solution.

15 Claims, 5 Drawing Figures RECORDER COLORIME'TER PATENTED DEC] 1 1975 saw 1 or 2 Fl G COLORIMETER RECORDER TEMP CONT FIG 2o ELUTING TIME FIG. 2b

ELUTING TIME INVENTORS YosHmRo ARlKAwA 515m TAKEUCHI HI AYUKI SAGUSA ntewbQZJ 4- w ATTORNEYS Craig,

METHOD OF AUTOMATICALLY ANALYZING AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLOR-DEVELOPING SOLUTION THEREFOR BACKGROUND OF THE INVENTION The present invention relates to a method of automatically analyzing amino acids by liquid chromatography and a new ninhydrin reagent for automatic amino acid analysis.

Most of the automatic amino acid analyzers now commercially available are based on a colorimetric method to determine the amino acid concentration after column chromatography, whereupon the fractions containing the amino acid eluted from the column are mixed with the ninhydrin reagent and subjected to the color developing reaction in a teflon tube with an internal diameter of 0.6-1 .0 mm equipped in the reaction cell and the colored sample solution is thenpassed to a flowing colorimeter wherein the optical density is measured and recorded.

Though the mechanism of the color developing reaction by the ninhydrin reagent has not thoroughly been clarified, the Schiff base intermediate theory as shown in equations (1 and (2) is rather significant at present.

Another reaction mechanism due to Ruhemann et al. involves hydrindantin, the reduced form of ninhydrin as the intermediate, as shown below:

l RCHCOOH RCI-IO +1fHa CO: (3)

The hydrindantin intermediate theory has been considered less convincingly than other theories represented by the above-mentioned Schiff base intermediate theory since the rate of coloration by a primary amino or an amino acid is faster than that by ammonia in the presence of excess hydrindantin. Therefore hydrindantin is regarded merely as playinga role as a stabilizer for the reaction intermediate; namely protecting the reaction intermediate from oxidative decomposition by the co-existing oxygen or other oxidizing agents.

The fact that the intensity of coloration increases with the concentration of hydrindantin in the ninhydrin color developing reaction of amino acid, other reducing agents which are unable to produce hydrindantin can not increase the intensity of coloration at all irrespective of the concentration thereof are explained conveniently by the hydrindantin intermediate theory.

Table 1 shows the composition of a conventional ninhydrin reagent employed for the automatic amino acid analyzer.

Table l Acetate Buffenl mole/liter (pH 5.5) Ninhydrin:0.l mole/liter Methylcellosolve:% SnCl 2I-l O:l.8 X 10 mole/liter The above-described conventional ninhydrin reagent employs stannous chloride (SnC1 as the reducing agent to produce hydrindantin by reducing a part of ninhydrin.

Though sodium (or potassium) cyanide has been reported to be used as the reducing agent instead of stannous chloride, it is not widely used because it is poisonous,except for a special case, and stannous chloride is still commonly adopted for an ordinary case.

It is effective to increase the concentration of hydrindantin for the purpose of enhancing the intensity of coloration, the amount of stannous chloride has to be increased consequently.

However, the solubility of the tin salt (Sn or Sn) becomes a significant matter when the concentration of the organic solvent, as well as the pH value of the test solution is high.

Heating the sample solution to l00C. in order to increase the reaction velocity in the case of using the conventional ninhydrin reagent generally produces deposition of a small amount of tin, which is considered to be the principal cause of choking the reaction coil.

Consequently, it is difficult to increase the amount of stannous chloride to be added to the test solution for the purpose of increasing the concentration of hydrindantin whenever the conventional ninhydrin reagent is employed.

According to the recent Schiff base intermediate theory, implying that hydrindantin is not compulsorily necessary but instead, the presence of a reducing agent being capable of stabilizing the reaction intermediate is efficient enough to afford the color developing reaction, an oxalate, sulfide, hydroquinone and other reducing agents which are inert to ninhydrin have been investigated. However, none of these agents showed effectiveness on adding significant amounts.

The reducing agents capable of affording hydrindantin in the reduction include an alkali metal, sodium sulfide, a chromite, hydrazine and the like, in addition to the conventionally known stannous chloride.

The reducing agent to be employed practically for the above purpose should not be positive to the ninhydrin reaction, it must have a greater solubility in the reaction mixture with the composition as shown in Table 1. Further, it should be stable, and easy to handle. For example, sodium sulfide is difficult to weigh owing to its hydroscopic property. l-Iydrazine itself is negative to the ninhydrin reaction; however, an amine which is decomposition product, is positive to the color developing reaction. The chromite ion and metallic sodium are not suitable as the reducing agent in view of instability and difficulty of handling.

SUMMARY OF THE INVENTION One of the objects of the present invention is to provide a new ninhydrin reagent for liquid chromatography with higher sensitivity of coloration and less trouble of choking the reaction coil compared with the conventional reagents.

Another object of the present invention is to provide a ninhydrin reagent which can be readily regenerated after deterioration. i

The general requirements of the reducing agent for the ninhydrin reagent are high purity, cheapness, greater solubility in the reagent solution, no harmful reaction for the sensitivity as well as other coloration, inertness to the equipment, and no deposition during use. Advantageously, after extensive investigations and a survey of a desired reducing agent, the present inventors have found that ascorbic acid is most promising for the purpose mentioned above.

The present invention provides a ninhydrin reagent solution for automatic amino acid analysis comprising ninhydrin, an organic solvent, a buffer solution, and ascorbic acid as the reducing agent.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be further appreciated by reference to the accompanying drawings, wherein:

FIG. 1 is a block diagram of an automatic amino acid analyzer employed in the present invention;

FIGS. 2a and 2b are graphs showing the change of coloration with the ninhydrin reagents according to the present invention and that by the Moore-Stein method;

FIG. 3 is a graph showing the rates of coloration with the ninhydrin reagents of the present invention and the Moore-Stein method respectively; and

FIG. 4 is a graph showing the stability of coloration with respect to time passed afforded by the ninhydrin reagent of the present invention, and the change of coloration upon further addition of ascorbic acid.

DETAILED DESCRIPTION OF THE INVENTION Ascorbic acid has been known to have a greater solubility to the organic solvent than the conventionally employed stannous chloride, and a potent reducing property. It has not yet been adopted i or the reducing agent for the ninhydrin reaction in spite of the excellent reducing property against ninhydrin, because ascorbic acid forms brown colored decomposition matters upon heating it alone. This increases the blank value of the colorimetric estimation of amino acids, causing a noise drift of the base line of the automatically recorded chromatogram.

Despite this possible disadvantage of ascorbic acid, the present inventors have discovered that when ascorbic acid is added to the ninhydrine reagent solution, the ascorbic acid yields oxalic acid and threonic acid which do not produce brown colored decomposition products.

The composition of the ninhydrin reagent according to the present invention is exemplified by the following tabulation indicated in Table 2.

Table 2 Acetate bufferrl mole/liter (pH 5.5) Ninhydrin:O.l mole/liter Methyl cellosolve: volume Ascorbic acid:0.2 X 10 0. 5 X 10* mole/liter It will be appreciated that in the ninhydrin reagent of this invention the methyl cellosolve may comprise from 60 to volume with the balance essentially comprising the acetate buffer, e.g. acetic acid-sodium acetate. Also, the range of ninhydrin may be from 0.05 to 0.2 mole/liter and the pH of the buffer solution may be from 5.4 to 5.6.

Ascorbic acid releases the hydrogen of two hydroxy groups present in the molecule to effect the reduction.

Thus because of particularly potent reducing capability and great solubility of ascorbic acid, necessary amount of hydrindantin can easily be produced on conversion of ninhydrin by adding ascorbic acid into the ninhydrin reagent solution.

Nevertheless the solubility of ascorbic acid is considerably great. The concentration of ascorbic acid is limited at about 10 mole/liter in actual use because the solubility of hydrindantin formed in the reaction of ascorbic acid with ninhydrin is about 10 mole/liter, and such excess of the hydrindantin becomes difficult to dissolve in methyl cellosolve; in particular, on mixing the buffer solution, the concentration of methyl cellosolve decreases, inducing the deposition of hydrindantin.

Ascorbic acid dissolves in the ninhydrin reagent up to the concentration of approximately 1 mole/liter. However, a low amount of the hydrindantin formed on addition of l X 10' mole/liter of ascorbic acid remains insoluble in 75% methylcellosolve.

Further, in practical use for an automatic usual amino acid analyzer, the concentration of methylcellosolve becomes lower than 25% in view of mixing of the buffer solution. The amount of hydrindantin should be confined at lower concentration. Thus the practical amount of ascorbic acid for the usual case has been found to be 0.5 X 10 mole/liter.

The minimum amount of ascorbic acid, namely 0.2 X mole/liter, compared with 1.8 X 10' mole/liter stannous chloride in Moore-Steins method gives rise to the same intensity of coloration as reflected by the absorbance. The increase of the absorbance given by amino acids reaches the maximum at the concentration level of 0.6 X 10' mole/liter, with respect to ascorbic acid, and at 0.8 X l0 m ole/liter, the absorbance be comes 100%.

The ninhydrin reagent is liable to oxidation, and on exposure to the air, it is rapidly oxidized and loses its activity. Therefore, it should be preserved in an atmosphere of nitrogen. Despite the fact that this reagent is inevitably oxidized by the oxygen of the air diffusing through teflon tubes, thereby causing the intensity of coloration to be lowered impairing the reproducibility of quantitative estimation, the duration of preservation of the Moore-Steins ninhydrin reagent wherein stannous chloride has been used as the reducing agent and that by the present invention is approximately the same, i.e. nearly about days. The reagent according to the present invention is characterized by recovery of the coloring capacity after the deterioration due to the air oxidation by readdition of ascorbic acid, for example, by addition of 0.4 X 10 mole/liter of ascorbic acid, i.e., the amount initially employed or a slightly lesser amount, the coloring capacity is recovered to the original level.

The readdition of the reducing agent in the conventional Moore-Stein method, has been impossible on account of low solubility of stannous chloride.

The reagent according to the present invention can be used as the color reagent for other methods as well as the ordinary ion exchange separation technique.

For example, it can be applied to the metallic salt resin column utilizing the difference in the complexing capacity of amino acids with metallic ions such as Cu, Ni, Co, Zn, Cd and the like. The column contains an ion exchange resin having as ion exchange groups, sulfonic acid groups, carboxyl acid groups or imino-diacetic acid groups. This method of chromatography is called Ligand method and is disclosed in U.S. Pat. application Ser. No. 742,993 entitled Method of Separating Mixture by Liquid Chromatography filed May 14, 1968, now U.S. Pat. No. 3,630,681, which disclosure is incorporated herein by reference.

The following specific examples will explain the method and color developing solution of the invention.

FIG. 1 is a block diagram of the automatic amino acid analyzer to be used as an embodiment of the present invention, wherein the container 1 containing the eluted fraction (containing for example, the acetate buffer) is connected to the metal salt form resin column 3 by the pump 2. The color reagent container 4 is connected under the column 3 through pump 5; the front of the crossing connecting tubes of the both being joined to the teflon tube 7 with a 0.3 to 0.8 mm internal diameter in the reaction cell. The teflon tube 7 is, in turn, connected to the flowing colorimeter 8, from which the solutionafter the determination is discharged.

The flowing colorimeter 8 is attached with the recorder 9 whereon the absorbance curve is plotted on a recording paper according to the absorbance of the solution. The device 10 is a heater and reference numeral 11 designates a thermistor, and 13 designates a stirrer.

FIGS. 2a and 2b show the elution curves on automatic analysis of amino acids wherein (A) is given by Moore-Steins color reagent and (B) by the reagent of the present invention, the both reagents gave approximately the same intensity of coloration.

FIG. 3 shows the change of the intensity of the coloration yielded by the reagent of the present invention and that of the Moore-Stein method in which the concentration of stannous chloride was 1.8 X 10' mole/liter. The color reagent according to the present invention (using ascorbic acid as the reducing agent) gave the same intensity of coloration as the Moore- Stein reagent at 0.2 X 10 mole/liter of ascorbic acid; whereas the absorbance reached to about 100% at 0.8 x 10 mole/liter with respect to ascorbic acid, though the ascorbic acid concentration up to 1.2 X 10' mole/liter is shown in the figure.

In FIG. 3 curvesl, 2, 3 and 4 indicate, respectively, the intensities of coloration given by glutamic acid, hydroxy-proline, sarcosine, and urea in relation to the amount of ascorbic acid used in accordance with this invention. The amount of stannous chloride in the Moore-Stein reagent was 1.8 X 10 mole/liter.

Though ascorbic acid can be added up to the precipitation of the resulting hydrindantin, the solubility of the latter should be taken in mind on mixing the eluted fractions with the ninhydrin reagent whereupon the concentration of methylcellosolve as the solvent for hydrindantin is decreased.

In addition, the absorbance reaches the constant value after the addition of a definite amount of ascorbic acid, whereas the blank value increases with the increase of the amount of ascorbic acid added, ascorbic acid should be added appropriately within the compensating capacity of the auto-analyzer.

FIG. 4 shows the relation between the periodical change of ninhydrin reagent using ascorbic acid as the reducing agent with the peak area of the absorbance curve. Though the ninhydrin reagent is sealed in the vessel in the stream of nitrogen, it deteriorates due to the air-oxidation provoked by the air diffusing through the stopper and the teflon tubes. I

The diffusion of the air into the vessel depends upon the structure of the stopper and the thickness of the teflon tubes, the deterioration characteristics of the Moore-Steins ninhydrin reagent and the reagent of the present invention are regarded the same provided other conditions are the same. In FIG. 4, the change of the intensity of coloration is indicated by the peak area of the recorded chromatogram on continuous use of the ninhydrin reagent containing 0.4 X 10 mole/liter of ascorbic acid in the presence of nitrogen. No change can be observed after 2 weeks (point A). The coloration becomes nil (point B) after aeration to allow the reagent to deteriorate completely by the oxidation. The intensity of coloration recovers to the original level (point C) by addition of 0.4 X 10 mole/liter of ascorbic acid after removal of remaining oxygen by blowing nitrogen gas into the solution.

As described above, a new ninhydrin reagent according to the present invention is devoid of precipitation at a high temperature and yields excellent color devel oping efficiency reflected in the absorbance. Further, by addition of a relatively large amount of ascorbic acid, the rate of the color developing reaction can be promoted which enables saving the length of the reaction tube.

In addition, the deteriorated reagent can be regenerated and becomes reusable by readdition of ascorbic acid.

In the above-described automatic amino acid analysis by liquid chromatography, the elute solution comprises 0.055 mole/liter of acetic acid buffer solution for pH of 4.1, 0.4 X 10' mole/liter of zinc and 8% by volume of ethanol.

The ninhydrin solution comprises one volume of ninhydrin and two volumes of acetic acid buffer.

In each run the flow rate of the mixture of above solutions in the ion exchange column with an 0.8 mm diameter and a length of 25 meters (heated to l C) is 135 cc per hour.

In all analyses the conditions were the same with the exception that 0.4 X 10 mole/liter of ascorbic acid was added to the above-noted ninhydrin solution in those runs conducted in accordance with the present invention.

In the column a strong acid type sulfonic acid polystyrene resin having 8% of cross linking ratio was used as ion exchange resin in all runs.

While the novel principles of the invention have been described, it will be understood that various omissions,

modifications and changes in these principles may be made by one skilled in the art without departing from the spirit and scope of the invention.

We claim:

1. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing amino acids together with an eluting solution through a separator column packed with ion exchange resins, separating the amino acids into their respective amino acid components by differences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to a means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to a recording means, the improvement wherein ascorbic acid is used as the reducing agent, said acid being added in an effective amount for converting a part of ninhydrin into hydrindantin.

2. A method according to claim 1, wherein the organic solvent is methylcellosolve.

3. The method for automatically analyzing amino acids according to claim 1, wherein the amount of ascorbic acid ranges between 0.2 X 10' and 0.5 X 10* mole per liter of said color developing solution.

4. The method for automatically analyzing amino acids according to claim 3, wherein about 100% absorbance is reached when ascorbic acid is used in a concentration of about 0.8 X 10' mole per liter.

5. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing an eluting solution containing a buffer agent and the same metal ions as those adsorbed on ion exchange resins in a separator column thereby to bring the column into an equilibrium, adding an amino acid sample to the separator column thereby to separate amino acids into the respective amino acid components by migration speeds due to differences in abilities to form complex salts of the respective amino acids with the metal ions, mixing an effluent eluate solution leaving the column with a color-developing solution containing ninhydrin, its reducing agent, and an organic solvent in an amount of 60 to by volume, heating the resulting mixutre solution in a reactor to carry out a colordeveloping reaction, continuously passing the resulting colored substance including Ruheman purple to a flow cell, where light is projected onto the solution and light absorption by the colored substance is effected, detecting a quantity of light, converting the detected quantity of light to electric signals and recording the electric signals, and the reaction system within the analytical system being isolated from surrounding atmosphere, the improvement wherein said reducing agent is ascorbic acid added in an effective amount for converting a part of ninhydrin into hydrindantin.

6. A method for automatically analyzing amino acids according to claim 5, wherein about absorbance is reached when ascorbic acid is used in a concentration of about 0.8 X 10 mole per liter.

7. A method for automatically analyzing amino acids according to claim 5, wherein the amount of ascorbic acid ranges between 0.2 X 10 and 0.5 X 10 mole/liter.

8. A method according to claim 5, wherein the organic solvent is methylcellosolve.

9. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acid by differences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to a means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to a recording means, the improvement wherein the reducing agent used is ascorbic acid and the color-developing solution, when deteriorated by oxidation, is recovered by adding ascorbic acid to said solution in order to convert a part of ninhydrin into hydrindantin.

10. A method according to claim 9, wherein the organic solvent is methylcellosolve.

11. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acids by differences in migration speeds of the amino acid in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to recording means, the improvement wherein the color-developing solution, when deteriorated by air oxidation, is recovered by adding ascorbic acid to said solution in an amount effective to convert a part of ninhydrin into hydrindantin.

12. A method according to claim 11, wherein the organic solvent is methylcellosolve.

13. A method according to claim 11, wherein the amount of ascorbic acid ranges between 2Xl0 and 0.5Xl0 mole/liter.

14. A ninhydrin color-developing solution for use in 15. A solution according to claim 14, wherein the automatically analyzing amino acids by liquid chromaamount f ascorbic acid ranges between -a and tography which comprises ninhydrin, as organic 501- 0 5X10 2 mole/liter vent, a buffer and ascorbic acid as a reducing agent for ninhydrin in an amount of 0.2 X to 0.5 X 10 5 mole per liter. 

2. A method according to claim 1, wherein the organic solvent is methylcellosolve.
 3. The method for automatically analyzing amino acids according to claim 1, wherein the amount of ascorbic acid ranges between 0.2 X 10 3 and 0.5 X 10 2 mole per liter of said color developing solution.
 4. The method for automatically analyzing amino acids according to claim 3, wherein about 100% absorbance is reached when ascorbic acid is used in a concentration of about 0.8 X 10 3 mole per liter.
 5. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing an eluting solution containing a buffer agent and the same metal ions as those adsorbed on ion exchange resins in a separator column thereby to bring the column into an equilibrium, adding an amino acid sample to the separator column thereby to separate amino acids into the respective amino acid components by migration speeds due to differences in abilities to form complex salts of the respective amino acids with the metal ions, mixing an effluent eluate solution leaving the column with a color-developing solution containing ninhydrin, its reducing agent, and an organic solvent in an amount of 60 to 80% by volume, heating the resulting mixutre solution in a reactor to carry out a color-developing reaction, continuously passing the resulting colored substance including Ruheman purple to a flow cell, where light is projected onto the solution and light absorption by the colored substance is effected, detecting a quantity of light, converting the detected quantity of light to electric signals and recording the electric signals, and the reaction system within the analytical system being isolated from surrounding atmosphere, the improvement wherein said reducing agent is ascorbic acid added in an effective amount for converting a part of ninhydrin into hydrindantin.
 6. A method for automatically analyzing amino acids according to claim 5, wherein about 100% absorbance is reached when ascorbic acid is used in a concentration of about 0.8 X 10 3 mole per liter.
 7. A method for automatically analyzing amino acids according to claim 5, wherein the amount of ascorbic acid ranges between 0.2 X 10 3 and 0.5 X 10 2 mole/liter.
 8. A method according to claim 5, wherein the organic solvent is methylcellosolve.
 9. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acid by differences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substAnces, leading the resulting solution containing said colored substances continuously to a means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to a recording means, the improvement wherein the reducing agent used is ascorbic acid and the color-developing solution, when deteriorated by oxidation, is recovered by adding ascorbic acid to said solution in order to convert a part of ninhydrin into hydrindantin.
 10. A method according to claim 9, wherein the organic solvent is methylcellosolve.
 11. In a method for automatically analyzing amino acids by liquid chromatography, which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acids by differences in migration speeds of the amino acid in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to recording means, the improvement wherein the color-developing solution, when deteriorated by air oxidation, is recovered by adding ascorbic acid to said solution in an amount effective to convert a part of ninhydrin into hydrindantin.
 12. A method according to claim 11, wherein the organic solvent is methylcellosolve.
 13. A method according to claim 11, wherein the amount of ascorbic acid ranges between 2 X 10 3 and 0.5 X 10 2 mole/liter.
 14. A ninhydrin color-developing solution for use in automatically analyzing amino acids by liquid chromatography which comprises ninhydrin, as organic solvent, a buffer and ascorbic acid as a reducing agent for ninhydrin in an amount of 0.2 X 10 3 to 0.5 X 10 2 mole per liter.
 15. A solution according to claim 14, wherein the amount of ascorbic acid ranges between 2 X 10 3 and 0.5 X 10 2 mole/liter. 